Characterization of Nuclear Protein Sp110a

• Main Authors: Wei-Syun Huang, 黃湋勛
• Other Authors: 顏伯勳
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/01496595487733397214

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 98 === Tuberculosis (TB) is one of the most important infectious diseases in the world. According to the epidemiological statistics, one-third of the world population is estimated to be infected with Mycobacterium tuberculosis (MTB), cause approximatedly 8 million new cases of tuberculosis globally each year , and result in about 2 million TB-related deaths annually. The majority of individuals infected with Mycobacterium tuberculosis remain asymptomatic and noninfectious, and only 10% will progress to active tuberculosis. There are many factors involved in the risk of individual infection and development of TB, and these include the interaction of the host body and it''s pathogens, stress, malnutrition, concomitant infections (for example HIV) and senescence. Evidence indicate that the susceptibility of host to MTB is associated with host immunity and genetic variation. Until now, the relationship between host resistance to TB and genetic variation have remained unclear. In previous studies, a genetic locus on mouse chromosome 1,named sst1 (supersusceptibility to tuberculosis 1), can control host resistance and susceptibility to tuberculosis. In further analysis, within the sst1 locus a gene designated Intracellular pathogen resistance 1(Ipr1). Ipr1 was upregulated in macrophages resistant to MTB but was not in the sst1 susceptible microphages. Therefore, Ipr1 gene may participate in innate immunity in mouse model of MTB infection and in the cellular regulatory mechanism of disease onset. The Sp110 nuclear body protein (Sp110) gene is the closest human homologue of the Ipr1 mouse gene.Sp110 proteins have 3 magor isoforms: Sp110a, Sp110b and Sp110c.The alternative splicing makes different isoforms of Sp110; however, the difference of roles and functions between these isoforms are still unclear. Thus, in this study, we investigated the characteristics and functional differences among the isoforms of Sp110 proteins. For this purpose, we constructed the eGFP-SP110 expression vector and co-transfect with four lentiviral structural gene to 293T cell line to generate lentiviral paricles containing our target gene. Then, we used dual-promoter lentiviral system to transduce human THP1 cell line to generate a stable clone, in which the expression of eGFP-Sp110a was controlled by the Tet-on system. After treated with doxycycline, the expression of eGFP-Sp110a protein can be induced. In the previous study, we found that the expression of eGFP-Sp110b/c protein was increased after treated with IFN, probably through the increase of protein stability. Thus, we will determine whether Sp110a has the same characteristics as SP110b/c. In addition, by coimmunoprecipitation, we found that some proteins binding with Sp110a/b/c protein were pulled down with an anti-eGFP antibody, including OAS1(2,5-oligoadenylate synthetase 1). The proteins interacting with eGFP-Sp110a/b/c will be further characterized. We will identify the proteins interacting with Sp110a by MS/MS analysis and compare the protein complex profile with that of Sp110b/c.